Complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact N-glycoprotein profiling reveals unconventional Asn71-glycosylation of human neutrophil cathepsin G

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Abstract

Neutrophil cathepsin G (nCG) is a central serine protease in the human innate immune system, but the importance of its N-glycosylation remains largely undescribed. To facilitate such investigations, we here use complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact glycoprotein profiling to accurately establish the micro- and macro-heterogeneity of nCG from healthy individuals. The fully occupied Asn71 carried unconventional N-glycosylation consisting of truncated chitobiose core (GlcNAcβ: 55.2%; Fucα1,6GlcNAcβ: 22.7%), paucimannosidic N-glycans (Manβ1,4GlcNAcβ1,4GlcNAcβ: 10.6%; Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ: 7.9%; Manα1,6Manβ1,4GlcNAcβ1,4GlcNAcβ: 3.7%, trace level of Manα1,6Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ), and trace levels of monoantennary α2,6- and α2,3-sialylated complex N-glycans. High-resolution/mass accuracy LC-MS profiling of intact nCG confirmed the Asn71-glycoprofile and identified two C-terminal truncation variants at Arg243 (57.8%) and Ser244 (42.2%), both displaying oxidation of solvent-accessible Met152. Asn71 appeared proximal (~19 Å) to the active site of nCG, but due to the truncated nature of Asn71-glycans (~5–17 Å) we questioned their direct modulation of the proteolytic activity of the protein. This work highlights the continued requirement of using complementary technologies to accurately profile even relatively simple glycoproteins and illustrates important challenges associated with the analysis of unconventional protein N-glycosylation. Importantly, this study now facilitates investigation of the functional role of nCG Asn71-glycosylation.
Original languageEnglish
Pages (from-to)1832-1854
Number of pages23
JournalBiomolecules
Volume5
Issue number3
DOIs
Publication statusPublished - Sep 2015

Bibliographical note

Copyright the Author(s) 2015. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

Keywords

  • neutrophil
  • cathepsin G
  • N-glycan
  • glycopeptide
  • glycoprotein
  • chitobiose
  • paucimannose
  • N-acetylglucosamine
  • glycomics
  • azurophilic granule
  • Glycomics
  • Cathepsin G
  • Glycoprotein
  • Azurophilic granule
  • Chitobiose
  • Glycopeptide
  • Neutrophil
  • Paucimannose

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